Multiparametric cell identification and sorting method and associated device

ABSTRACT

The invention relates to an method of analysing biological particles, in particular to be conducted in a cell sorter, with following steps: placing the particles to be analysed into a carrier flow, carrying out a first analysis of the particles moving with the carrier flow, selecting at least one particle in dependence on the result of the first analysis, carrying out a second analysis of the selected particle in a decelerated condition. Furthermore, the invention comprises a corresponding analysis device.

The invention relates to a method for analysing preferably biologicalparticles, in particular for analysing biological cells in a cellsorter, according to claim 1, as well as to a corresponding analysingdevice according to claim 14.

From Müller, T. et al.: “A 3-D microelectrode system for handling andcaging single cells and particles”, Biosensors and Bioelectronics 14(1999) 247-256, a method for analysing biological cells is known, inwhich method the cells to be analysed are suspended in a carrier flow ofa microfluidic system and are dielectrophoretically manipulated andsorted. In the carrier flow, the cells to be analysed are first lined upby a funnel-shaped dielectrophoretic electrode arrangement, and thenheld in a dielectrophoretic cage so that the cells located in the cagecan be analysed in their resting state, for which purpose microscopic,spectroscopic or optical fluorescence analysis methods can be used.Depending on the analysis of the cells trapped in the dielectrophoreticcage, these cells can subsequently be sorted, for which purpose anoperator controls a sorting device comprising a dielectrophoreticelectrode arrangement which is arranged in the carrier flow downstreamof the dielectrophoretic cage.

The above-described known method for analysing cells is associated witha disadvantage in that the cells to be analysed are often very differentin a sample. In the case of greatly heterogeneous samples, from whichfor example certain target cells are to be identified by a method, withthese target cells then having to be isolated, the target cells oftenaccount for only a small fraction of the entire sample. The other cellsdo not have the desired characteristics or are no longer vital, i.e.they are already dead. Furthermore, it often happens that the cells arenot completely singled out, but instead that many cells pass through thesystem as aggregations of two or more cells. This is an undesirableresult. However, detailed analysis of individual cells or aggregationsin a field cage is a time-consuming process so that analysis of theentire cell sample in the field cage would take a very long time.

It is thus the object of the invention to improve the above-describedknown method for analysing cells such that analysis of biological cellsor cell agglomerations that are of no interest (e.g. dead cells) in thedielectrophoretic cage can be avoided.

Starting from the known method for analysing cells, as described in theintroduction, this object is met by the features of claim 1, or—inrelation to a corresponding analysing device—by the features of claim14.

The invention comprises the general technical teaching according towhich, prior to analysing in the dielectrophoretic cage, the particlessuspended in the carrier flow are first subjected to a preliminaryanalysis of the particles moving with the carrier flow so that theparticles of interest for further analysis can subsequently be trappedand analysed in the dielectrophoretic cage.

The preliminary investigation can for example relate to the intensity ofa fluorescence, the vitality of a cell and/or the question of whether asingle cell or an aggregation is involved. Furthermore, during thepreliminary investigation it can be determined whether cells ormaterials are involved which in shape and size are not the primaryobjective of closer analysis, for example impurities or other cells,provided they differ from the target cells.

Thus in the method according to the invention for analysing cells firsta preliminary analysis of the particles suspended in the carrier flowand a selection of certain particles take place depending on the resultof the preliminary analysis, while the actual principal analysis is onlycarried out in relation to the previously selected particles which forthis purpose are decelerated so as to make possible a meaningfulprincipal analysis which would be made more difficult if the particleswere moving.

Within the scope of the invention it is not mandatory for the particlesselected depending on the preliminary analysis to be completely broughtto a halt prior to the principal analysis, for example by trapping theseparticles in an dielectrophoretic cage. Instead, within the scope of theinvention it is also possible for the particles selected depending onthe preliminary analysis to be decelerated in the particle stream onlyto such an extent that a meaningful analysis of the particles becomespossible.

Furthermore, it should be mentioned that in the context of thisinvention the term “particle” is to be understood in a general senserather than being limited to individual biological cells. Furthermore,this term also includes synthetic or biological particles, whereinparticular advantages arise if the particles are biological materials,for example biological cells, cell groups, cell components orbiologically relevant macromolecules, each if applicable in associationwith other biological particles or synthetic carrier particles.Synthetic particles can comprise solid particles, liquid particles,particles delimited from the suspension medium, or multiphase particleswhich form a separate phase in relation to the suspension medium in thecarrier flow.

Preferably, the particle selected depending on the preliminary analysisand analysed in more detail in the context of the principal analysis issorted and/or treated depending on the result of the principal analysis.For example, in the principal analysis various cell types can bedifferentiated and subsequently can be sorted accordingly. It is howeveralso possible for the particles selected in the context of thepreliminary analysis to be manipulated by dielectrophoretic elementsdepending on the result of the principal analysis, wherein thedielectrophoretic elements described in the above-mentioned publicationof Müller, T. et al. can be used.

Within the context of the preliminary analysis, for example, atransmitted-light analysis, fluorescence analysis and/or impedancespectroscopy can be carried out. However, in the preferred embodiment ofthe invention first a transmitted-light analysis is carried out,followed by a fluorescence analysis, wherein the transmitted-lightanalysis and the fluorescence analysis preferably take place inspatially separated regions of interest. The transmitted-light analysiscan for example allow a differentiation between living and deadbiological cells, while fluorescence analysis can be used to investigatewhether the particles suspended in the carrier flow carry a fluorescencemarker.

If within the scope of the preliminary analysis both a transmitted-lightanalysis and a fluorescence analysis are carried out in spatiallyseparated regions of interest, it is advantageous if the region ofinterest for the transmitted-light analysis is situated in the carrierflow upstream of the region of interest for the fluorescence analysis.However, as an alternative it is also possible for the region ofinterest for the transmitted-light analysis to be arranged in thecarrier flow downstream of the region of interest for the fluorescenceanalysis.

Preferably, within the scope of the preliminary analysis of theparticles moving with the carrier flow an optical image is taken, whichmakes possible digital image evaluation for classifying the particles.Preferably, in this process the particles are morphologically analysed,for example to make it possible to differentiate a single biologicalcell from a cell agglomeration. The term “optical image” used in thecontext of the present description is however to be interpreted in ageneral sense and is not limited to two-dimensional images in thetraditional sense of the term. Instead, in the context of the presentinvention the term “optical image” also includes point-shaped orline-shaped optical scanning of the carrier flow or of the particlessuspended in the carrier flow. For example, the brightness along a lineacross the carrier flow channel can be superintegrated for the purposeof detecting and classifying individual particles.

In a transmitted-light analysis the differentiation between living anddead cells can take place by evaluating the intensity distribution inthe optical image taken. For example phase-contrast illumination is aspecial principle of such a transmitted-light analysis. Intransmitted-light analysis living biological cells have an annularstructure wherein the margin is relatively bright and the centre isdarker, while dead biological cells are approximately uniform inbrightness and appear dark against the background.

In the principal analysis of the particles it is for example possible tolocate certain molecules within a cell. For example, in the context ofthe principal analysis it is possible to locate, within a cell,molecules that are marked with a fluorescent dye. The fluorescent dyecan for example comprise molecular-biologically produced tags of greenfluorescent protein and its derivatives, other autofluorescent proteins.However, fluorescent dyes which establish a covalent or non-covalentbond with a cellular molecule are also suitable as fluorescent dyes.Furthermore, fluorigenic substances can also be used as fluorescentdyes, which fluorigenic substances are converted by cellular enzymes tofluorescent products or so-called FRET pairs (fluorescence resonanceenergy transfer). The state of the fluorescent dyes used can for examplebe differentiated by means of their spectral characteristics or by meansof bioluminescence.

By means of locating molecules within a cell it is also possible todetermine the structure and function of the molecules. It is for examplepossible to differentiate between their presence in the plasma membrane,in the cytosol, in the mitochondria, in the Golgi apparatus, inendosomes, in lysosomes, in the nucleus, in the spindle apparatus, inthe cytoskeleton, co-localisation with actin, tubulin.

Furthermore, within the context of the principal analysis and/or thepreliminary analysis the morphology of a cell can be determined. In thisprocess it is also possible to use dyes.

Moreover, within the context of the principal analysis and/or thepreliminary analysis two or more states of a cell population can bedifferentiated.

Furthermore, it is possible within the context of the principal analysisto determine a cellular signal by means of translocation of afluorescence-marked molecule, e.g. receptor activation followed byreceptor internalisation; receptor activation followed by the binding ofarrestin; receptor aggregation; transfer of a molecule from the plasmamembrane to the cytosol, from the cytosol to the plasma membrane, fromthe cytosol to the nucleus, or from the nucleus to the cytosol.

Furthermore, it is also possible within the context of the principalanalysis and/or the preliminary analysis to determine the interactionbetween two molecules, wherein preferably at least one of theinteracting molecules carries a fluorescence marker, and the interactionis for example shown by collocation of two fluorescent dyes, a FRET or achange in the fluorescence lifetime.

However, it is also possible within the context of the principalanalysis and/or the preliminary analysis to determine the status of acell within a cell cycle, wherein preferably the morphology of the cellor the colouration of the cellular chromatin is evaluated.

A further option in relation to the principal analysis and/or thepreliminary analysis consists of determining the membrane potential of acell, wherein preferably membrane-potential-sensitive dyes are used.Preferably, for this purpose dyes are used which are sensitive inrelation to the plasma membrane potential and/or the mitochondrialmembrane potential.

Moreover, it is also possible within the context of the principalanalysis and/or the preliminary analysis to determine the vitality of acell, wherein preferably the morphology of the cell is evaluated and/orfluorigenic substances are used which can differentiate between livingand dead cells.

Furthermore, in the principal analysis and/or the preliminary analysisit is also possible to analyse cytotoxic effects and/or determine theintracellular ph values.

It is also possible within the context of the principal analysis and/orthe preliminary analysis to determine the concentration of one orseveral ions within a cell.

During the principal analysis and/or the preliminary analysis it is alsopossible to determine any enzymatic activity within a cell, whereinpreferably fluorigenic substances or chromogenic substances, inparticular kinases, phosphatases or proteases can be used.

Moreover, it is also possible during the principal analysis and/or thepreliminary analysis to determine the production performance of cellsthat produce biological products such as for example proteins, peptides,antibodies, carbohydrates or fats, wherein one of the described methodscan be used.

Finally, within the context of the principal analysis it is alsopossible to determine cell stress paths, metabolic paths, cell growthpaths, cell division paths and other signal transduction paths.

Furthermore, the invention relates to a corresponding analysing devicefor implementing the above-described method for analysing cells.

The analysing device according to the invention preferably comprisesoptics in order to take an image of the particles.

Preferably, the optics of the analysing device according to theinvention are adjustable to make it possible to set the magnification,the focus and/or the field of vision, or to select a particular opticalfilter, wherein adjustment of the optics can take place by an actuator(e.g. an electric motor).

It has already been mentioned above that deceleration of the particlespreferably takes place by a dielectrophoretic cage, which is known perse. However, in one embodiment of the invention the dielectrophoreticcage is not only used for decelerating the suspended particles for adetailed investigation, but it also functions as a switch or adistribution switchpoint in that the suspended particles, depending onthe detailed analysis in the cage, are fed to one of several outletlines. To this effect, the individual electrodes of thedielectrophoretic cage are preferably selectable independently of eachother. Furthermore, to this effect the dielectrophoretic cage ispreferably arranged at the branch point of the output lines.

Moreover, a funnel-shaped electrode arrangement can be arranged in oneor several of the output lines so as to prevent sinking of the suspendedparticles in the outlet lines. This is advantageous because the carrierflow in the output lines has a speed profile which shows only a slowflow speed near the wall so that sinking of the particles in the outletlines could lead to deposits near the wall.

Furthermore, there is the option of supplying the suspended particles byway of two separate carrier flow lines which flow into a common carrierflow line. In this arrangement a dividing wall can be arranged in thecommon carrier flow line, in the region of the mouth of the two carrierflow lines, which dividing wall in the common carrier flow lineseparates two separate partial flows, wherein the two partial flows canbe analysed. Depending on the result of this analysis, the particlessuspended in the two partial flows can then be brought together. Theparticles brought together can then in the above-described manner befixed in a dielectrophoretic cage and can be subjected to detailedanalysis. Finally, the cells released from the dielectrophoretic cagecan then be fed to one of several outlet lines, depending on the resultof the detailed analysis.

The invention is particularly advantageous in that cells can be analysedin aseptic conditions or conditions with few germs and can be isolatedaccordingly.

Other advantageous improvements of the invention are characterised inthe dependent claims or are explained below with reference to thefigures, in the context of the description of the preferred embodimentsof the invention. The following are shown:

FIG. 1 a fluidic diagram of a cell sorter comprising a sorter chip,according to the invention;

FIG. 2 the carrier flow channel of the sorter chip with severaldielectrophoretic elements;

FIG. 3 a diagrammatic representation of the analysing optics of the cellsorter of FIG. 1;

FIG. 4 a diagram to explain the differentiation between dead and livingbiological cells;

FIGS. 5 a-5 e an example of the method for analysing cells, according tothe invention, in the form of a flow chart; and

FIGS. 6-9 alternative embodiments of the carrier flow channel of thesorting chip with several dielectrophoretic elements.

The diagram of FIG. 1 shows a cell sorter according to the invention,which cell sorter dielectrophoretically sorts biological cells by meansof a microfluidic sorting chip 1.

The techniques of dielectrophoretically influencing biological cellshave for example been described in Müller, T. et al.: “A 3-Dmicroelectrode system for handling and caging single cells andparticles”, Biosensors and Bioelectronics 14 (1999) 247-256, so that nodetailed description of the dielectrophoretic processes in the sortingchip 1 is provided below, but instead in this regard reference is madeto the above-mentioned publication.

For fluidic contacting, the sorting chip 1 comprises several connections2-6, wherein fluidic contacting of the connections 2-6 is described inDE 102 13 272, whose contents shall form part of the presentdescription.

The connection 2 of the sorting chip 1 is used to accommodate a carrierflow with the biological cells to be sorted, while the connection 3 ofthe sorting chip 1 is used to lead away the selected biological cellswhich are not further analysed on the sorting chip 1. The selectedbiological cells can be collected by a suction injector 7 that can beconnected to the connection 3 of the sorting chip 1. In contrast to theabove, the outlet 5 of the sorting chip 1 is used to lead away thebiological cells that are of interest, which biological cells cansubsequently be processed or analysed.

Furthermore, the connections 4 and 6 of the sorting chip 1 are used tosupply a so-called enveloping flow, whose task it is to lead theselected biological cells to the connection 5 of the sorting chip 1. Asfar as the function of the enveloping flow is concerned, reference ismade to the German patent application DE 100 05 735 so that in thepresent document there is no need to provide a detailed description ofthe function of the enveloping flow.

The connections 4 and 6 of the sorting chip are connected by way of twoenveloping flow lines 8, 9, a Y-piece 10 and a four-way valve 11 to apressure vessel 12 in which there is a cultivation medium for theenveloping flow.

The pressure vessel 12 is pressurised by way of a compressed air line 13so that the buffer solution in the pressure vessel 12 (e.g. acultivation medium) with a corresponding position of the four-way valve11 flows to the connections 4, 6 of the sorting chip 1 by way of theY-piece 10 and the enveloping flow lines 8, 9.

However, as an alternative, the enveloping flow can also be implementedby principles other than through the pressure vessel 12 with the buffersolution, for example using an injector pump or a peristaltic pump.

In contrast to the above, the connection 2 of the sorting chip 1 isconnected to a particle injector 15 by way of a carrier flow line 14.

Upstream, the particle injector 15 is connected by way of a T-piece 16to a carrier flow injector 17, which is manually driven and injects apredetermined liquid flow of a carrier flow.

Furthermore, upstream, the T-section 16 is connected to a three-wayvalve 20 by way of a further four-way valve 18 and an enveloping flowline 19. The three-way valve 20 makes it possible to flush theenveloping flow lines 8, 9 and the carrier flow line 14 prior to actualoperation.

To this effect the three-way valve 20 is connected upstream by way of aperistaltic pump 21 to three three-way valves 22.1-22.3, to which ineach case an injector reservoir 23.1-23.3 is connected. In thisarrangement the injector reservoirs 23.1-23.3 are used to supply a fillflow for flushing the entire fluidic system prior to actual operation,wherein the injector reservoir 23.1 contains e.g. 70% ethanol while theinjector reservoir 23.2 preferably contains distilled water as a fillflow substance. The injector reservoir 23.3 contains e.g. a buffersolution as a fill flow substance.

Furthermore, the cell sorter comprises a collecting vessel 27 for excessenveloping flow, as well as a collecting vessel 28 for excess fill flow.

Below, first the flushing process is described which is carried outprior to the actual operation of the cell sorter to free the envelopingflow line 8, 9, the carrier flow line 14 and the remaining fluidicsystem of the cell sorter of any air bubbles and impurities.

To this effect first the three-way valve 22.1 is opened, and ethanolfrom the injector reservoir 23.1 is injected as a filler flow, whereinthe peristaltic pump 21 first conveys the ethanol to the three-way valve20. The ethanol is thus used to reduce the number of germs in the system(so as to establish an aseptic analysis and selection process) and alsoto completely displace any air from the fluidic system.

During the flushing process the three-way valve 20 is set such that partof the fill flow conveyed by the peristaltic pump 21 is conveyed by wayof the fill flow line 19 while the remaining part of the fill flowconveyed by the peristaltic pump 21 reaches the four-way valve 11. Thetwo four-way valves 11, 18 are again set such that the fill flow isconveyed through the enveloping flow lines 8, 9 and the carrier flowline 14. Furthermore, cultivation medium flows from the pressure vessel12 into the collecting vessel 27 in order to briefly flood the lines.

Following flushing of the cell sorter with ethanol, as described above,flushing with distilled water or a buffer solution is carried out in thesame way, wherein in each case the three-way valves 22.2 or 22.3 areopened.

In the flushing process described above, the four-way valve 18 can leadaway excess fill flow to the collecting vessel 28.

After the flushing process the three-way valves 22.1-22.3 are closed andthe peristaltic pump 21 is switched off.

In order to initiate the sorting operation the four-way valve 11 is setsuch that the pressure vessel 12 is connected to the Y-piece 10 so thatthe cultivation medium in the pressure vessel 12 is pushed into theenveloping flow lines 8, 9 as a result of the overpressure in thepressure vessel 12.

Furthermore, during the sorting operation the four-way valve 18 isadjusted such that there is no flow connection between the T-piece 16and the four-way valve 18.

The carrier flow injected by the carrier flow injector 17 then flows byway of the T-piece 16 into the particle injector 15, wherein a furtherinjector 29 injects biological cells into the carrier flow. Subsequentlythe carrier flow with the injected biological cells flows from theparticle injector 15 by way of the carrier flow line 14 to theconnection 2 of the sorting chip.

Furthermore, it should be mentioned that a temperature sensor 30 hasbeen fitted to the particle injector 15 so as to measure the temperatureT of the particle injector 15.

Furthermore, both on the particle injector 15 and on the receptacle forthe sorting chip 1 there is a temperature control element 31 in the formof a Peltier element so that the particle injector 15 and the sortingchip 1 can be heated or cooled.

In this arrangement, the heating energy or cooling energy Q is specifiedby a temperature controller 32 which on the inlet side is connected tothe temperature sensor 30 and which controls the temperature T of theparticle injector 15 to a predefined desired value.

Below, with reference to FIG. 2 a carrier flow channel 33 is describedwhich is arranged in the sorting chip 1 of the cell sorter, wherein saidcarrier flow channel 33 branches into two outlet lines 34, 35, whereinoutlet line 34 is connected to connection 5 of the sorting chip 1 and isused for conveying positively selected particles, while outlet line 35is connected to connection 3 of the sorting chip 1 and serves to removethe selected particles.

In the carrier flow channel 33, downstream of the connection 2 of thesorting chip 1, a funnel-shaped dielectrophoretic electrode arrangement36 is arranged whose task it is to line up, in sequence one behindanother in the carrier flow channel 33, the particles suspended in thecarrier flow. The precise design and the function of the electrodearrangement 36 are described in the publication, mentioned in theintroduction, by Müller T. et al., wherein the contents of saidpublication shall form part of the present description so that belowthere is no need to provide a detailed description of the electrodearrangement 36.

Downstream of the electrode arrangement 36, a dielectrophoretic cage 37is arranged in the carrier flow channel 33, which dielectrophoretic cage37 makes it possible to trap the particles suspended in the carrier flow33 and to fix said particles in a region of interest UF for in-depthanalysis. As far as the design and function of the dielectrophoreticcage 37 is concerned, reference is again made to the cited publicationby Müller T. et al., so that there is no need to provide a detaileddescription in this respect.

Downstream of the dielectrophoretic cage 37, in a branch region of thecarrier flow channel 33, there is a sorting device which comprises adielectrophoretic electrode arrangement 38, wherein as far as the designand function of the electrode arrangement 38 is concerned, reference isalso made to the publication by Müller T. et al. cited in theintroduction. The electrode arrangement 38 sorts the particles suspendedin the carrier flow either into the outlet line 34 or into the outletline 35, wherein the selection is carried out depending on a principalanalysis carried out on the particles fixed in the cage 37, as will bedescribed in detail below.

Furthermore, in the branch region of the carrier flow line 33 a flowguide device is arranged which also comprises a dielectrophoreticelectrode arrangement 39 and whose task it is to prevent any reverseflow of particles from the outlet line 35 to the outlet line 34. To thiseffect the electrode arrangement 39 is v-shaped and comprises two legs,wherein one leg of the electrode arrangement 39 protrudes into theoutlet line 34 while the other leg of the electrode arrangement 39protrudes into the outlet line 35.

Below, with reference to FIGS. 2 and 3, a description is provided of theway the particles suspended in the carrier flow are analysed in thesorting chip 1.

In the context of a preliminary analysis of the particles, first atransmitted-light analysis is carried out in one region of interestROI1, and a fluorescence analysis is carried out in a further region ofinterest ROI2, wherein ROI1 is arranged in the carrier flow channel 33so as to be upstream of the region of interest ROI2 for fluorescenceanalysis.

Both transmitted-light analysis and fluorescence analysis are carriedout by the detection unit D, diagrammatically shown in FIG. 3, whichdetection unit D for the purpose of image acquisition comprises a CCDcamera 40, which is arranged downstream of the sorting chip 1 and isaligned towards a deviation mirror 41.

Above the sorting chip 1 a light emitting diode 42 is arranged as alight source for transmitted-light analysis, wherein between the lightemitting diode 42 and the sorting chip 1 a condenser 43 is arranged,which can for example comprise a phase contrast diaphragm.

Below the sorting chip 1, in the optical path of the condenser 43, alens 44 is arranged.

In the case of a transmitted-light analysis the CCD camera 40 takes animage of the region of interest ROIL by way of the deviation mirror 41and the lens 44.

Furthermore, the detection unit D comprises several electric motordriven actuators 45.1-45.3, which make it possible to adjust the lens44, the filter block 47 and the deviation mirror 41. Changing the lens44 makes it possible to change the magnification and the focus. Incontrast to this, the filter block 47 can be adjusted to selectdifferent filters. Adjusting the deviation mirror 41 serves the purposeof shifting the field of vision along the carrier flow channel 33 sothat any deposits in the carrier flow channel 33 can be detected.

For the purpose of excitation of fluorescence during the fluorescenceanalysis, the detection unit D comprises a light source 46 (e.g. alaser), which by way of a filter block 47 makes possible excitation offluorescence of the biological cells suspended in the carrier flow line33, wherein the CCD camera 40 takes a corresponding fluorescence image.

Below, the various forms of biological cells appearing in thetransillumination image are described with reference to FIG. 4. Theupper region of FIG. 4 shows a living cell 48 and a dead cell 49, andthe lower region shows the associated intensity gradients 50, 51 in thetransillumination image. This shows that the living cell 48 has arelatively dark nucleus, while the interior of the dead cell 49 isilluminated evenly. This difference makes it possible to differentiatebetween a living cell 48 and a dead cell 49, as will be described indetail below.

Below, the method, according to the invention, for analysing cells isdescribed with reference to the flow chart shown in FIGS. 5 a to 5 e.

At the beginning of the method first the carrier flow line 14 and theenveloping flow lines 8, 9 are flushed with a 70% ethanol solution, thenwith distilled water and finally with a buffer solution so as to cleanthe fluidic system of the cell sorter and in particular so as to free itof any air bubbles and impurities.

After this, the carrier flow is injected into the carrier flow line 14from the carrier flow injector 17, wherein, after the enveloping flowhas been supplied, the biological cells to be analysed are injected intothe carrier flow by the injector 29 on the particle injector 15 asdescribed below.

Furthermore, the cultivation medium contained in the pressure vessel 12for the enveloping flow is pushed, by the compressed air supplied by wayof the compressed air line 13, from the pressure vessel 12 into theenveloping flow lines 8, 9 which lead to the connections 4 or 6 of thesorting chip 1 and which support further transfer of the particlesselected in the sorting chip 1 by way of connection 5 of the sortingchip 1.

In the carrier flow channel 33 of the sorting chip 1, the suspendedparticles are first aligned, one behind the other in the direction offlow, by the electrode arrangement 36, as is diagrammatically shown by adashed arrow.

Subsequently, in the region of interest ROI1, several phase contrastimages B₁, . . . , B_(n) are taken in succession in order to determinethe movement speed of the suspended particles and to differentiatebetween living cells and dead cells, as will be described in detailbelow.

In order to determine the movement speed of the suspended particles, foreach of the phase contrast images B₁, . . . , B_(n) an intensity signalI₁, . . . , I_(n) is determined in that the image intensity in the phasecontrast images B₁, . . . , B_(n) is superintegrated by columns, i.e. ata right angle in relation to the direction of flow. In other words, theindividual intensity signals I₁, . . . , I_(n) have a signal peak at thelocation of a biological cell, wherein a signal peak between theintensity signals I₁, . . . , I_(n) is shifted in accordance with themovement speed of the cells and the time interval between the intensitysignals I₁, . . . , I_(n).

Subsequently, a cross correlation function φ_(i) is calculated forsubsequent intensity signals I_(i), I_(i+1). Calculating the crosscorrelation function φ_(i) serves to determine the movement speed of thecells in the carrier flow channel 33 of the sorting chip 1 so that thedielectrophoretic cage 37 can be selected at the right point in time totrap a particular cell.

Subsequently, the maximums are calculated for the individual crosscorrelation functions φ_(i) (x) depending on the displacement x inlongitudinal direction of the carrier flow channel 33.

The movement speed v of the cells in the carrier flow channel 33 resultsas a quotient from the average value of the maximums of the crosscorrelation functions and the time interval between subsequent phasecontrast images B₁, . . . , B_(n).

The movement speed v of the cells can be used within the context offeedback for pump control, i.e. for checking whether the calculated pumprate agrees with the actual pump rate and to what extent anyreadjustment may have to take place. In particular, the movement speed vcan be used to detect whether there are any malfunctions in the system,on the basis of which malfunctions the cells flow too slowly (blockage),are immobile, or even flow backward. All these malfunctions can bedetected and remedied in this way, e.g. by flushing the system.

However, as an alternative, the above-described determination of themovement speed v of the cells can also take place outside the regions ofinterest ROI1, ROI2. Basically, cell tracking within the entire carrierflow channel 33 or within any desired regions of the carrier flowchannel 33 is possible.

Furthermore, the signal shape of the intensity signals I₁, . . . , I_(n)provides information about the size of the particles and any aggregateformation. Overall, evaluation of the intensity signals is important forcontrolling and automating the entire unit, namely the pumps, thedielectrophoretic electrode elements (e.g. when does caging take placeand when does switching take place), detailed image capture in the cage37, and sample storage.

In a further step the point in time of trapping t_(F) is calculated, atwhich point in time the cage 37 has to be selected in order to trap theanalysed particle for the subsequent principal analysis in the region ofinterest UF. The point in time of trapping t_(F) simply results from themovement speed v of the particle and the distance from the cage 37.

Moreover, in a further step, the particle spacing d_(P) betweenneighbouring particles is determined. This is important fordifferentiating between an individual cell and a cell agglomeration, aswill be described in detail below.

Below, the process sequence described in FIG. 5 c is explained, in whichprocess sequence differentiation between dead cells and live cells takesplace. To this effect, cell margin points x₁, x_(r) are determined inwhich the intensity in the phase contrast image exceeds a predefinedthreshold value I_(TH).

Subsequently, a check is made whether there is an intensity minimumbetween the cell margin points x₁, x_(r). If this is the case and aminimum intensity is present, then this cell is a living cell, as isshown in FIG. 4. Otherwise the cell is classified as being dead, inorder to then carry out a respective selection, as will be described indetail below.

Following the above-described differentiation between dead cells andliving cells, in the process sequence shown in FIG. 5 d the luminance Lof the individual cells is determined in that the intensity I of a cellbetween the cell margin groups x₁ and x_(r) is superintegrated.

Afterwards the luminance L, determined in this way, of the cell iscompared to a minimum value L_(min) and a maximum value L_(max).

If the determined luminance of the cell is within this region, thetransmitted-light illumination is switched off and the excitation offluorescence by way of the light source 46 is switched on. After this, afluorescence image is taken in the region of interest ROI2, and thefluorescence I_(F) of the cell is measured.

However, it is also possible to have the excitation of fluorescenceswitched on permanently, wherein only the transmitted-light illuminationis switched off if the determined luminance of the cell is within theabove-mentioned region of interest.

If the measured fluorescence I_(F) of the cell exceeds a predeterminedlimiting value I_(min) this indicates that the cell concerned has afluorescence marker.

In contrast to the above, if the measured fluorescence I_(F) is belowthe predefined limiting value I_(min) it can be assumed that the cellconcerned does not carry a fluorescence marker.

In the process sequence shown in FIG. 5 e particular cells are thenselected, wherein the differentiation between living cells and deadcells as well as the check for any fluorescence marker is taken intoaccount. For example, it is possible to select those cells that areliving and carry a fluorescence marker, whereas other cells aredeselected.

In a further step, there is a differentiation between individual cellson the one hand and cell aggregation on the other hand, in that thepreviously determined particle spacing d_(P) is compared to a predefinedminimum value d_(MIN). If the minimum value d_(MIN) is not reached, itis assumed that the particle is a cell aggregate, so that the process isterminated. In contrast to this, if the particle spacing d_(P) exceedsthe predefined minimum value d_(MIN), it is assumed that the particle isan individual cell and the process is continued with the steps describedbelow.

At the predetermined point in time of trapping t_(F) the cells selectedin this way are then trapped in the dielectrophoretic cage 37 and arefixed in this way so that subsequently a principal analysis of thetrapped cell is possible at a higher resolution and a longer exposuretime.

The selected cells, i.e. as a rule the living cells that carry afluorescence marker, are then allowed by the electrode arrangement 38 toenter the outlet line 34, whereas the deselected cells (e.g. dead cells)are conveyed to the outlet line 35.

The principal analysis in the region of interest UF can involve imageswith excitation of fluorescence, wherein one or several excitationwavelengths can be used simultaneously or offset in time. To thiseffect, suitable dichroic mirrors are used in the filter block 47. Inthis arrangement the fluorescence light of one or several wavelengths issimultaneously channelled to one or several cameras. To this effectsuitable emission filter inserts are used in the filter block 47 orsuitable emission splitters are used. In this way it is possible toproduce, simultaneously or in sequence, images of the selected cell withthe use of several fluorescent dyes. Furthermore it is possible toproduce an image of the selected cell with white light phase-contrastillumination. This is necessary to detect whether one or severalnon-fluorescence-marked cells still adhere to a fluorescence-markedcell, which would lead to—normally undesirable—contamination of thissingle fluorescence-marked cell.

The embodiment shown in FIG. 6 largely corresponds to the embodimentshown in FIG. 2 so that, for the sake of avoiding repetition, referenceis made to the above description and, below, identical reference numbersare used for corresponding components, which reference numbers fordifferentiation have merely been marked with an apostrophe.

One characteristic of this embodiment consists of the simplerconstruction design of the dielectrophoretic electrode arrangement 36′arranged on the inlet side of the carrier flow channel 33′, whichelectrode arrangement 36′ lines up, in sequence one behind the other, inthe carrier flow channel 33′, the particles suspended in the carrierflow.

A further characteristic of this embodiment consists of a hook-shapedelectrode arrangement 52′, commonly referred to as a “hook”, beingarranged in the carrier flow channel 33′ downstream of the electrodearrangement 36′, with the function of this hook being to seize particlesand to quasi park them. The precise design and function of the electrodearrangement 52′ is for example described in Müller, T. et al.: “LifeCells in Cellprocessors” in Bioworld 2-2002 so that there is no need toprovide a detailed description of the electrode arrangement 52′ in thisdocument, wherein the contents of the above-mentioned printedpublication shall to the full extent form part of this description.

In the carrier flow channel 33′ there is a region of interest 53′between the electrode arrangement 52′ and the dielectrophoretic cage 37′to carry out the preliminary analysis, described above in relation tothe regions of interest ROIL and ROI2.

In this arrangement, a further region of interest 54′ is located in thedielectrophoretic cage 37′ so that in the dielectrophoretic cage 37′ ananalysis of the decelerated particles can be carried out.

A further characteristic of this embodiment consists of a funnel-shapedelectrode arrangement 55′ being arranged in the outlet line 34′ for thepositively selected particles, with the function of said funnel-shapedelectrode arrangement 55′ corresponding to the function of the electrodearrangement 36′ and the task of the electrode arrangement 55′ being tocentre the particles in the outlet line 34′. This is advantageousbecause the particles in the outlet line 34′ have a tendency to sink andcan therefore settle near the wall where the flow speed is low. Theelectrode arrangement 55′ prevents such sinking of the particles and inthis way keeps the particles in the middle of the outlet line 34′ wherethe flow speed is at its maximum.

Furthermore, it should be mentioned that the electrode arrangements 36′,52′ and the dielectrophoretic cage 37′ are arranged off-centre in thecarrier flow line 33′. This results in the particles contained in thecarrier flow, when they are released from the dielectrophoretic cage37′, automatically reaching the outlet line 35′ for negatively selectedparticles if the electrode arrangement 38′ is not selected. Thisprovides an advantage in that the electrode arrangement 38′ needs to beselected only rarely if in the carrier flow only a few particles arecontained that are to be positively selected.

The alternative embodiment shown in FIG. 7 largely agrees with thepreviously described embodiment shown in FIG. 6 so that, for the sake ofavoiding repetition, reference is made to the previous description and,below, identical reference numbers are used for correspondingcomponents, which reference numbers for differentiation have been markedwith two apostrophes.

One characteristic of this embodiment consists of the dielectrophoreticcage 37″ being arranged at that position in which the carrier flowchannel 33″ branches into the two outlet lines 34″, 35″. Moreover, theindividual electrodes of the dielectrophoretic cage 37″ can be selectedseparately so that the dielectrophoretic cage 37″ can carry out twofunctions, namely firstly the function of a cage, and secondly thefunction of a switch or a distribution switchpoint. Thedielectrophoretic cage 37″ can thus fix the particles in the carrierflow not only for analysis in the region of interest 54″ but also feedthe particles to one of the two outlet lines 34″, 35″.

The term “branch point” used in the context of the present descriptionis to be understood in a general sense rather than being limited to thegeometric intersection point of the outlet lines. Instead, it is alsopossible for the cage 37″ or the distribution switchpoint to be arrangedupstream of the intersection point of the outlet lines. For example, theterm “branch point” also includes the so-called “separatrix”, i.e. theseparation line of the laminar flow in the carrier flow channel.

Furthermore, in this arrangement and in the following embodiments theelectrode arrangements 36″, 52″, the cage 37″ and the measuring stations53″, 54″ are arranged at the centre of the carrier flow channel 33″.

The embodiment shown in FIG. 8 largely agrees with the embodimentdescribed above and shown in FIG. 7 so that, for the sake of avoidingrepetition, reference is made to the previous description and, below,identical reference numbers are used for corresponding components, whichreference numbers for differentiation have been marked with threeapostrophes.

One characteristic of this embodiment consists of the construction ofthe dielectrophoretic cage 37′″ having only six spatially arrangedelectrodes, wherein the individual electrodes can be selected separatelyso that the cage 37′″ can act as a switch or distribution joint or as acage, as desired.

Finally, FIG. 9 shows a further embodiment of a possible arrangement ina sorting chip. In this arrangement two carrier flow lines 56, 57 leadinto a common carrier flow line 58, wherein the respectively suspendedparticles are supplied by way of the two carrier flow lines 56, 57.

A funnel-shaped electrode arrangement 59, 60 is arranged in each of thetwo carrier flow lines 56, 57 so as to centre the particles contained inthe carrier flows of the two carrier flow lines 56, 57.

Upstream in the common carrier flow channel 58, at the point where thetwo carrier flow lines 56, 57 join, there is a dividing wall 61 so thatthe particles suspended in the carrier flows of the two carrier flowlines 56, 57 are first guided in the carrier flow line 58 parallel sideby side and separately of each other.

In the region of the dividing wall 61 in the carrier flow line 58 thereare two regions of interest 62, 63 in order to subject the suspendedparticles to a preliminary analysis when they flow past, wherein thepreliminary analysis can for example be carried out in the mannerpreviously described in the context of FIG. 2.

Downstream of the two regions of interest 62, 63 there is afunnel-shaped electrode arrangement 64 in the carrier flow line 58,wherein said funnel-shaped electrode arrangement 64 centres theparticles suspended in the two partial flows on both sides of thedividing wall 61 and feeds said particles to a dielectrophoretic cage 65which can fix the particles for analysis in a further region of interest66.

Downstream behind the dielectrophoretic cage 65 there is a furtherelectrode arrangement 67, which after release by the cage 65 feeds theparticles suspended in the carrier flow depending on the result of theanalysis in the region of interest 66 to any one of three outlet lines68, 69, 70. In this arrangement the outlet lines 68, 70 are used to leadaway the negatively selected particles, while the outlet line 69 is usedfor onward conveying of the positively selected particles. The electrodearrangement 67 thus has to be actively selected if particles are to beconveyed into the outlet lines 68, 70 for the negatively selectedparticles, while in contrast to this, no selection takes place for thepositively-selected particles. This arrangement is thereforeparticularly suited to those analyses where only few particles arenegatively selected.

The invention is not limited to the preferred embodiments describedabove. Instead, a multitude of variants and modifications is possiblewhich also utilise the inventive step and thus fall within the scope ofthe patent.

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 29. A methodfor analyzing particles, in particular for biological particles, withthe following steps: placing the particles to be analysed at least inone carrier flow; carrying out at least one first analysis of theparticles moving with the carrier flow; selecting at least one particledepending on the result of the first analysis; decelerating the selectedparticle; and carrying out at least one second analysis of the selectedparticle in the decelerated state.
 30. The method for analyzing cellsaccording to claim 29, comprising at least one of the steps of sortingand treating the selected particles depending on the result of thesecond analysis.
 31. The method for analyzing cells according to claim29, wherein within the scope of at least one of the first analysis andthe second analysis at least one of a transmitted-light analysis and afluorescence analysis is carried out.
 32. The method for analyzing cellsaccording to claim 31, wherein the transmitted-light analysis is carriedout in a first region of interest and the fluorescence analysis iscarried out in a second region of interest, wherein the two regions ofinterest are spatially separate from each other.
 33. The method foranalyzing cells according to claim 29, wherein within the scope of thefirst analysis at least one optical image of the particles is taken. 34.The method for analyzing cells according to claim 29, wherein within thescope of the first analysis at least one of an electrical andelectromagnetic analysis is carried out.
 35. The method for analyzingcells according to claim 34, within the scope of the first analysis animpedance analysis is carried out.
 36. The method for analyzing cellsaccording to claim 29, wherein during the first analysis and the secondanalysis the particles are analysed morphologically or in relation totheir size.
 37. The method for analyzing cells according to claim 29,wherein during at least one of the first analysis a check is madewhether the particles in the carrier flow comprise a single biologicalcell or several biological cells, wherein such particles which consistof a single biological cell are selected for the second analysis. 38.The method for analyzing cells according to claim 29, wherein during thefirst analysis a check is made whether the particles in the carrier floware living cells or dead cells.
 39. The method for analyzing cellsaccording to claim 38, wherein within the scope of the first analysis atransmitted-light analysis of the particles is carried out, wherein anoptical image of the particles is taken, and the intensity distributionin the image of the particles is evaluated.
 40. The method for analyzingcells according to claim 29, wherein during the first analysis a checkis made, by means of a fluorescence analysis, as to whether theparticles in the carrier flow exceed a specified threshold for afluorescence marker.
 41. The method for analyzing cells according toclaim 29, further for the purpose of deceleration comprising at leastone of the steps of fixing the particle that has been selected in thefirst analysis in a field cage and slowing down the carrier flow.
 42. Ananalyzing device for analyzing particles, in particular biologicalparticles, comprising a carrier flow channel for accommodating a carrierflow with the particles suspended therein; a first measuring station forcarrying out a first analysis of the particles moving with the carrierflow; a selection unit for selecting particles depending on the resultof the first analysis; wherein the selection unit comprises adeceleration device for decelerating the selected particles; and asecond measuring station for carrying out a second analysis of theselected particles in the decelerated state.
 43. The analyzing deviceaccording to claim 42, wherein the second measuring station is arrangeddownstream of the first measuring station.
 44. The analyzing deviceaccording to claim 42, wherein downstream of the second measuringstation at least one of a treatment device and a sorting device isarranged in order to treat or sort the selected particles depending onthe result of at least one of the first and the second analysis.
 45. Theanalyzing device according to claim 44, wherein the carrier flow channeldownstream of the second measuring station branches into at least twoflow channels, wherein the sorting device is arranged in the branchregion of the carrier flow channel.
 46. The analyzing device accordingto claim 45, wherein the sorting device comprises a dielectricdistribution switchpoint which is arranged in the branch region of thecarrier flow channel.
 47. The analyzing device according to claim 45,wherein in the branch region of the carrier flow channel a flow guidedevice is arranged in order to prevent reverse flow of the carrier flowor of the particles from one of the two flow channels into the otherflow channel.
 48. The analyzing device according to claim 47, whereinthe flow guide device comprises an electrode.
 49. The analyzing deviceaccording to claim 48, wherein the electrode of the flow guide device isessentially v-shaped and comprises two legs which essentially extend inthe direction of the two branching-off flow channels.
 50. The analyzingdevice according to claim 42, wherein the deceleration device comprisesa dielectric cage.
 51. The analyzing device according to claim 42,wherein focusing electrodes are arranged in the carrier flow channelupstream of the first measuring station.
 52. The analyzing deviceaccording to claim 42, wherein at least one of the first measuringstation and the second measuring station comprises optics for taking animage.
 53. The analyzing device according to claim 52, wherein theoptics are movable in order to displace the image at least along thecarrier flow channel.
 54. The analyzing device according to claim 53,wherein for displacing the image, the optics are connected to anelectromechanical actuator.
 55. Method of using an analyzing deviceaccording to claim 42 in at least one of medical or pharmaceuticalresearch, diagnostics and forensic medicine.
 56. Method of using ananalyzing device according to claim 42 for separating various celltypes, for example in particular apoptic and necrotic cells, cells withdifferent expression patterns and/or stem cells.
 57. The method foranalyzing cells according to claim 30, wherein within the scope of atleast one of the first analysis and the second analysis at least one ofa transmitted-light analysis and a fluorescence analysis is carried out.58. The method for analyzing cells according to claim 57, wherein thetransmitted-light analysis is carried out in a first region of interestand the fluorescence analysis is carried out in a second region ofinterest, wherein the two regions of interest are spatially separatefrom each other.